Dissociated cell cultures of dopaminergic neurons from embryonic rat mesencephalon were grown in completely defined serum-free medium. Survival and growth of the neurons were assessed by quantifying the level of the dopamine uptake system. Under these restrictive growth conditions, the cultures develop over a long period in vitro particularly from day 14 to days 21-23. To study the role of soluble growth factors during this period, culture medium was replaced with fresh medium with or without added factors. Replacement of medium with fresh medium with or without factors led to the complete destruction of the cultures. This damage was blocked with low levels of a competitive (APV) or non-competitive (Mk-801) antagonist of the NMDA-type of glutamate receptors. An antagonist of non-NMDA receptors (CNQX) was ineffective. Excitatory amino acids (EAA) operating via NMDA receptors are responsible for culture damage. While (mu)M levels of glutamate or glutamate agonists can damage the cultures, the fresh medium nominally contains no glutamate. Susceptibility of the cultures to damage following medium change appears abruptly between day 13 and 16 in vitro and probably reflects the expression of NMDA receptors at this time. Change of medium on or before day 13 in vitro does not cause destruction of the cultures. However, this early medium change does have an effect on the cells; when the medium on these cultures is changed at later times, they are resistant to the destruction that normally occurs. The induction of the resistant state appears to involve non-NMDA receptors. Interplay between cells expressing various subtypes of EAA.